JP2018510158A - Viral therapy using antibody combinations - Google Patents

Abstract

Disclosed herein are viruses that can be used in methods for the treatment of cancer. More specifically, the virus expresses two or more antibodies that induce an effective anti-tumor immune response. This virus can also be used in diagnostic methods. [Selection figure] None

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit and priority thereto of US Provisional Application No. 62 / 135,096, filed Mar. 18, 2015, the entire disclosure of which is incorporated herein by reference. Is.

  Cancer is the second most common cause of death in the United States after heart disease. In the United States, cancer accounts for a quarter of all causes of death. Among all cancer patients diagnosed in 1996-2003, the 5-year relative survival rate was 66%, an increase from 50% in 1975-1977 (Cancer Facts & Figures American Cancer Society: Atlanta, GA (2008)) ). Finding highly effective cancer treatments is the main goal of cancer research.

  The present invention relates generally to the treatment of human cancer and more specifically to the use of several treatment modalities combined to induce an effective anti-tumor immune response.

  As used herein, in some embodiments, a method for treating a solid tumor comprising a recombinant virus capable of infecting cells in the tumor or a virus capable of infecting cells in the tumor. Wherein the virus comprises the following: (i) two or more antibodies that target the tumor microenvironment (TME); (ii) two or more stimulatory or inhibitory targets that target TME Disclosed is a method of expressing a protein, or (iii) at least one antibody that targets TME and at least one protein that is stimulatory or inhibitory that targets TME. In some embodiments, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the virus is a replication competent oncolytic vaccinia virus (VACV). In some embodiments, the virus expresses two or more antibodies that target TME. In some embodiments, the two or more antibodies are selected from the group consisting of: (i) an antibody that binds to a protein that stimulates angiogenesis and / or angiogenesis; (ii) epidermal growth factor receptor. Antibodies that bind to receptor tyrosine kinases selected from the group consisting of the body (EGFR), Her2 / C-neu, Her3 and Her4, and (iii) antibodies that bind to proteins involved in the development of epithelial-mesenchymal interactions . In some embodiments, the two or more antibodies are selected from the group consisting of: an antibody that binds to vascular endothelial growth factor (VEGF), an antibody that binds to epidermal growth factor receptor (EGFR), And an antibody that binds to fibroblast activation protein (FAP). In some embodiments, one of the two or more antibodies is an antibody that binds to VEGF. In some embodiments, the antibody that binds to VEGF is G6-31. In some embodiments, one of the two or more antibodies is an antibody that binds to EGFR. In some embodiments, the antibody that binds to EGFR is anti-EGFR VHH. In some embodiments, one of the two or more antibodies is an antibody that binds to FAP. In some embodiments, the antibody that binds to FAP is M036. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to EGFR. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to FAP. In some embodiments, the two or more antibodies include an antibody that binds to EGFR and an antibody that binds to FAP. In some embodiments, the VACV is selected from GLV-1h444 and GLV-1h446. In some embodiments, the method further comprises administering an additional cancer therapy. In some embodiments, the additional cancer therapy is selected from the following: radiation therapy, chemotherapy, immunotherapy, phototherapy, and combinations thereof. In some embodiments, the tumor is selected from: glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumor, and melanoma. In some embodiments, the virus is delivered intravenously to the subject. In some embodiments, the virus is delivered intravenously directly to the tumor or is delivered to the subject in the region of the tumor. In some embodiments, the method further comprises providing the subject with at least one additional virus capable of infecting cells within the tumor, the small virus comprising: (i) the tumor microenvironment ( An antibody that targets TME) or (ii) a stimulatory or inhibitory protein that targets TME; wherein the at least one additional virus is (i) an antibody that targets the tumor microenvironment (TME) or ( ii) one of (i) an antibody targeting TME or (ii) a stimulating or inhibitory protein targeting TME that is different from that expressed by a virus expressing a stimulatory or inhibitory protein that targets TME Express one or more.

  Disclosed herein are, in some embodiments, recombinant viruses capable of infecting cells in solid tumors, wherein: (i) two or more that target the tumor microenvironment (TME) (Ii) two or more proteins that stimulate or inhibit TME, or (iii) at least one antibody that targets TME and at least one protein that stimulates or inhibits TME. The recombinant virus that expresses. In some embodiments, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the virus is a replication competent oncolytic vaccinia virus (VACV). In some embodiments, the virus expresses more than one antibody. In some embodiments, the two or more antibodies are selected from the group consisting of: (i) an antibody that binds to a protein that stimulates angiogenesis and / or angiogenesis; (ii) epidermal growth factor receptor. Antibodies that bind to receptor tyrosine kinases selected from the group consisting of the body (EGFR), Her2 / C-neu, Her3 and Her4, and (iii) antibodies that bind to proteins involved in the development of epithelial-mesenchymal interactions . In some embodiments, the virus comprises two or more heterologous nucleic acids that encode or express two or more antibodies selected from the group consisting of: binds to vascular endothelial growth factor (VEGF) Antibodies that bind to epidermal growth factor receptor (EGFR), and antibodies that bind to fibroblast activation protein (FAP). In some embodiments, one of the two or more antibodies is an antibody that binds to VEGF. In some embodiments, the antibody that binds to VEGF is G6-31. In some embodiments, one of the two or more antibodies is an antibody that binds to EGFR. In some embodiments, the antibody that binds to EGFR is anti-EGFR VHH. In some embodiments, one of the two or more antibodies is an antibody that binds to FAP. In some embodiments, the antibody that binds to FAP is M036. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to EGFR. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to FAP. In some embodiments, the two or more antibodies include an antibody that binds to EGFR and an antibody that binds to FAP. In some embodiments, the VACV is selected from GLV-1h444 and GLV-1h446. In some embodiments, the virus is a Lister strain. In some embodiments, the A34R gene is replaced with an A34R gene from another vaccinia virus strain. In some embodiments, the A34R gene is replaced with an A34R gene from vaccinia IHD-J strain. In some embodiments, the virus comprises a deletion of the A35R gene. In some embodiments, the virus further comprises an additional heterologous nucleic acid molecule that encodes a diagnostic or therapeutic protein. In some embodiments, the additional heterologous nucleic acid molecule encodes a diagnostic protein. In some embodiments, the diagnostic protein comprises a luciferase, a fluorescent protein, an iron storage molecule, an iron transporter, an iron receptor, a protein that binds to an iron receptor or a contrast agent, a chromophore, or a detectable compound or detectable ligand It is chosen from among them. In some embodiments, the additional heterologous nucleic acid molecule encodes a therapeutic protein. In some embodiments, the therapeutic protein is a cytokine, chemokine, immunomodulatory molecule, antigen, single chain antibody, antisense RNA, prodrug converting enzyme, siRNA, angiogenesis inhibitor, toxin, antitumor oligopeptide, Selected from among antimitotic proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics and tissue factor.

  Disclosed herein are, in some embodiments, host cells comprising a recombinant virus as disclosed herein.

  Disclosed herein are, in some embodiments, tumor cells that comprise a recombinant virus as disclosed herein.

  Disclosed herein is, in some embodiments, a mammalian organism that includes or is infected by a recombinant virus as disclosed herein.

  Further disclosed herein is the use of a recombinant vaccinia virus as disclosed herein for the treatment of a tumor in a subject.

  Disclosed herein is the use of a vaccinia virus, as disclosed herein, for the preparation of a pharmaceutical composition for the treatment of a tumor in a subject. In some embodiments, the pharmaceutical composition further comprises an anticancer compound.

  Also disclosed herein is the use of a recombinant virus that can infect cells in a tumor, or a cell comprising a virus that can infect cells in a tumor, in a method for treating a solid tumor in a subject. The method comprises administering to a subject a recombinant virus capable of infecting cells in a tumor, or a cell comprising a virus capable of infecting cells in a tumor, wherein the virus comprises: (i) a tumor Two or more antibodies that target the microenvironment (TME); (ii) two or more proteins that are stimulatory or inhibitory that target TME, and (iii) target at least one antibody and TME that target TME Expresses at least one protein that is stimulatory or inhibitory.

FIG. 2 illustrates schematic diagrams of antibodies and novel VACV. (A) Schematic representation of anti-EGFRVHHFLAG, anti-VEGF scAb (GLAF-2), and anti-FAP scAb (GLAF-5) constructs. (B) Genomic structure of the novel recombinant VACV and their parental virus along it. GLV-1h442 and GLV-1h282 were derived from GLV-1h68 by substituting the lacZ expression cassette at the J2R locus with anti-EGFRVHHFLAG and GLAF-5 cassettes, respectively, under the control of the PSEL promoter. GLV-1h164 replaces the lacZ expression cassette at the J2R locus with hNET under the PSE promoter and the GLV by replacing the gusA expression cassette at the A56R locus with the GLAF-2 cassette under the PSL promoter. Derived from -1h68. GLV-1h444 and GLV-1h446 were derived from GLV-1h164 by replacing the hNET expression cassette at the J2R locus with anti-EGFRVHHFLAG and GLAF-5 cassettes, respectively, under the control of the VACV PSEL promoter. All viruses contain a ruc-gfp expression cassette at the F14.5L locus. PSE, PSEL, PSL, P11, and P7.5 are VACV early, late / late, late, 11K, and 7.5K promoters, respectively. It demonstrates that individual therapeutic antibodies of viral expression targeting TME significantly enhance viral therapy in A549 and DU145 tumor xenograft models. (A) Antibodies targeting VEGF expressed from GLV-1h164 significantly enhanced viral therapy. Mice bearing A549 xenograft tumors (n ≧ 7) were treated with virus alone, Avastin alone, PBS alone or virus in combination with Avastin. When the tumor volume reached 450 mm 3, a single dose of virus (2 × 10 6 pfu / mouse) was given intravenously (iv). Avastin was administered i.p. at a dose of 5 mg / kg twice weekly for 5 weeks starting at 10 dpi. p. Administered. Arrows indicate the beginning and end of Avastin treatment. Statistical analysis was performed using one-way ANOVA (*** P <0.001, ** P <0.01, ** P <0.05). The asterisk indicates a comparison between the GLV-1h68 group, the GLV-1h164 group (black), and the GLV-1h68 + Avastin group (open). αV indicates anti-VEGF. (B) Antibodies targeting EGFR expressed from GLV-1h442 significantly enhanced viral therapy. Mice bearing A549 xenograft tumors (n ≧ 7) were treated with virus alone, Erbitux alone, PBS alone or virus in combination with Avastin. When the tumor volume reached 450 mm 3, a single dose of virus (2 × 10 6 pfu / mouse) was administered i. v. Gave in. Erbitux was administered i.p. at a dose of 3 mg / kg twice weekly for 5 weeks starting at 10 dpi. p was administered. Arrows indicate the beginning and end of erbitux treatment. Statistical analysis was performed using one-way ANOVA (** P <0.01, ** P <0.05). The asterisk indicates a comparison between the GLV-1h68 group and the GLV-1h442 group. αE indicates anti-EGFR. (C) Antibodies targeting FAP expressed from GLV-1h282 significantly enhanced viral therapy. When tumor volume reached 450 mm 3, mice bearing A549 xenograft tumors (n ≧ 7) were given a single dose of GLV-1h68 or GLV-1h282 (2 × 10 6 pfu / mouse) i. v. Injected. Statistical analysis was performed using one-way ANOVA (** P <0.01, ** P <0.05). An asterisk indicates a comparison between the GLV-1h68 group and the GLV-1h282 group. αF indicates anti-FAP. (D) When the tumor volume reached 450 mm 3, each VACV strain (2 × 10 6 pfu / mouse) was transferred to DU145 tumor-bearing mice (n = 5). v. Tumor volume was monitored weekly thereafter. Statistical analysis was performed using one-way ANOVA (*** P <0.001, ** P <0.01, ** P <0.05). The asterisk indicates a comparison between the GLV-1h68 group, the GLV-1h164 group (black), the GLV-1h282 group (outlined), and the GLV-1h442 group (grey). Illustrates the effect of antibodies expressed in tumors targeting VEGF, EGFP and FAP in TME. (A) Effect of virus treatment on tumor vasculature in DU145 tumor (n = 3). Sections were stained for CD31 expression (red). GFP expression (green) indicates viral infection. (B) Quantitative analysis of blood vessels was performed by counting CD31 + blood vessels in 8 non-overlapping microscopic fields per slide. Statistical analysis was performed using an unpaired two-sided Student's t test (** P <0.01, ** P <0.05). (C) Effect of virus treatment on cell growth in DU145 tumor (n = 3). Sections were stained for Ki67 expression (red). GFP expression (green) indicates viral infection. The scale bar corresponds to 1 mm for a and c. (D) Quantitative analysis of cell proliferation was performed by counting Ki67 + cells in 8 non-overlapping microscopic fields per slide. Statistical analysis was performed using an unpaired two-sided Student's t test (** P <0.01, ** P <0.05). (E) Immunohistochemical characterization of viral replication and stromal neogenesis. Formalin-fixed and paraffin-embedded tumor tissues (n = 4-5 per group) were cut into 5 μm sections and subjected to H & E staining. Adjacent sections were stained with anti-A27 for VACV, anti-CD31 for blood vessels, and anti-FAP for FAP + stromal cells. (F) Bar graph shows the average number of CD31 + cells and FAP + stromal cultures in infected or uninfected areas. CD31 + and FAP + stromal cultures in 5 non-overlapping microscopic fields (100 × magnification) per tumor were counted. Statistical analysis was performed using an unpaired two-sided Student's t test (** P <0.01, ** P <0.05). Illustrates how FaDu tumor growth was significantly inhibited by GLV-1h282 expressing an anti-FAP scab. However, FaDu tumors did not respond to treatment with GLV-1h68. Illustrates how each recombinant VACV expressed the intended antibody. Illustrates that the expression of the two antibodies did not negatively affect the effectiveness of viral replication. The virus replication assay was performed in A549 cells with a multiplicity of infection (MOI) of 0.01. Illustrate that two therapeutic antibodies expressed in virus that target TME further improve viral therapy. (A) Enhancement of therapeutic effect of GLV-1h444 in A549 tumor-bearing nude mice. Mice (n ≧ 7) were treated with virus alone, avastatin + erbitux, PBS alone, or virus in combination with Avastin and erbitux. When the tumor volume reached 450 mm 3, a single dose of virus (2 × 10 6 pfu / mouse) was administered i. v. Gave in. Avastatin and erbitux were administered i.p. at a dose of 5 mg / kg and 3 mg / kg twice a week for 5 weeks starting at 10 dpi. p was administered. Arrows indicate the beginning and end of avastatin and erbitux treatment. Statistical analysis was performed using one-way ANOVA (*** P <0.001, ** P <0.01, ** P <0.05). The asterisk indicates a comparison between the GLV-1h68 group, the GLV-1h444 group (black), the GLV-1h68 + Avastin + erbitux group (open), and the PBS + avastatin + erbitux group (grey). (B) Enhanced therapeutic effect of GLV-1h446 compared to its parent virus in A549 tumor-bearing nude mice. When the tumor volume reached 450 mm 3, each VACV strain was administered i.p. to mice (n ≧ 7) at a dose of 2 × 10 6 pfu / mouse. v. Injected. Statistical analysis was performed using one-way ANOVA (*** P <0.001, ** P <0.01, ** P <0.05). An asterisk indicates a comparison between the GLV-1h68 group and the GLV-1h446 group (black). (C) Enhanced therapeutic effect comparing GLV-1h444 and GLV-1h446 to their parental virus in DU145 tumor-bearing mice. When the tumor volume reached 450 mm 3, each VACV strain alone was administered i.m. to mice (n = 5) at a dose of 2 × 10 6 pfu / mouse. v. Injected. Statistical analysis was performed using one-way ANOVA (*** P <0.001, ** P <0.01, ** P <0.05). The asterisk indicates a comparison between the GLV-1h68 group, the GLV-1h444 group (black), and the GLV-1h446 group (outlined). (Df) Detection of changes in antibody and tumor volume in mouse serum in mice bearing A549 tumor after VACV treatment. Blood samples were collected from the retro-orbit at 7, 21, and 35 dpi. The concentration of antibody in mouse serum was determined by ELISA using precoated FAP, EGFR and VEGF plates. Tumors were measured with a digital caliper. Statistical analysis was performed using an unpaired two-sided Student's t test (*** P <0.001). Illustrates tumor growth curves of individual mice bearing A549 tumors. 2 illustrates the biodistribution of viruses in various organs and tumors in A549 tumor-bearing mice at 14 dpi as determined by standard virus plaque assays. 2 illustrates the evaluation of possible adverse effects of antibody-expressing VACV administration in mice. Assessment was performed by assessing net weight change over the course of treatment. In both A549 and DU145 tumor xenograft models, no significant change in average net body weight was observed in either the treatment group or the subject group. 2 illustrates that two virally expressed antibodies by GLV-1h444 and GLV-1h446 contribute to the suppression of cell proliferation in tumors. (A) Inhibition of cell proliferation by VACV in DU145 tumor (n = 3). GFP expression (green) indicates viral infection. Cell proliferation was examined by staining with anti-Ki67 antibody (red). (B, c) Quantitative analysis of cell proliferation in uninfected or infected areas was performed by counting Ki67 + cells in 8 non-overlapping microscopic fields per slide. Statistical analysis was performed using an unpaired two-sided Student's t test (** P <0.01, ** P <0.05). 2 illustrates that two virally expressed antibodies by GLV-1h444 and GLV-1h446 contribute to the suppression of angiogenesis in tumors. (A) Reduction of tumor vasculature by VACV in DU145 tumor (n = 3). Sections were stained with CD31 antibody (red). GFP expression (green) indicates viral infection. (B, c) Quantitative analysis of blood vessels in uninfected or infected areas was performed by counting CD31 + cells in 8 non-overlapping microscopic fields per slide. Statistical analysis was performed using an unpaired two-sided Student's t-test (*** P <0.001, ** P <0.01, ** P <0.05). The scale bar indicates 1 mm for a and d.

  Despite long advances in anticancer treatment over the past 30 years, cancer remains the leading cause of death worldwide. Overlooking the important role of the tumor microenvironment (TME) in cancer growth and metastasis may be one reason that no fully effective cancer treatment has yet been found. Angiogenesis and cellular hyperproliferation in the tumor stroma not only supports cancer growth, but also contributes to cancer development, eg, in metastasis.

  Factors within TME such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) play a critical role in the development and development of cancer. High level expression of VEGF or EGFR correlates with poor prognosis prediction in patients with breast cancer, colon cancer, lung cancer, head and neck cancer, and other cancers. An anti-VEGF monoclonal antibody (mAb), bevacizumab (Avastin), was approved in 2004 by the US Food and Drug Administration (FDA) for the treatment of metastatic colon cancer and subsequently other metastatic cancers. Also in 2004, the anti-EGFR mAb cetuximab (Erbitux) was also approved by the FDA for the treatment of metastatic colon cancer. However, the clinical efficacy of Avastin and erbitux is somewhat limited, probably due to poor tumor penetration and rapid clearance of mAbs derived from the circulation and is high over frequent intervals and long periods. It required administration of a dose, and the cost of treatment was quite high.

  Improvements in the pharmacodynamic properties of current mAb treatments and the identification of additional functions targeting TME would be of considerable benefit. For example, G6-31 is an improved anti-VEGF antibody derived from a phage display library that has better affinity and improved therapeutic efficacy in animal models than Avastin. To improve tumor penetration, a 15 kDa single domain antibody against llama-derived EGFR has recently been developed (referred to as anti-EGFR VHH). This llama Nanobody was non-immunogenic in mice and proved to block EGF binding to EGFR, thereby inhibiting EGFR signaling and exhibiting specific tumor targeting. Anti-EGFR VHH is used for molecular imaging and therapeutic applications. A highly conserved protein, FAP (also known as seprase) is abundantly expressed, particularly in the stroma of invasive cancers. High levels of FAP expression are associated with cancer progression. Species cross-reactive FAP-specific single chain antibody (scAb), M036, was isolated by serial phage display and directed to FAP on various human carcinoma stromal cells and mouse host stroma in human tumor xenografts. It was shown to bind. The therapeutic capacity of M036 has not yet been evaluated.

  Replication competent oncolytic vaccinia virus (VACV) GLV-1h68 settles and replicates and lyses in tumor cells in a human xenograft nude mouse model after administration of a single dose. In addition, the recombinant VACV may be genetically modified to express a functional transgene including scAb. VACV expressing the anti-VEGF scAb GLAF-1 designed by the sequence of G6-31 has previously been shown to significantly improve anticancer therapeutic efficacy in mice compared to the parental virus GLV-1h68. It was. This therapeutic efficacy was further enhanced in combination with radiation therapy.

  Therefore, a novel recombinant VACV expressing a novel TME targeted antiproliferative activity was constructed by encoding a scAb against FAP (GLV-1h282) and a single domain antibody against EGFR (GLV-1h442). VACV expressing these individual antibodies significantly suppresses tumor growth in a xenograft tumor model, thereby verifying the functionality and therapeutic activity of the virus-expressed antibody. Finally, an additional recombinant VACV encoding both antibodies with both anti-proliferative and angiogenesis-inhibiting activities targeting VEGF and EGFR (GLV-1h444) or VEGF and FAP (GLV-1h446) is generated It was done. A novel VACV expressing TME targeted antibodies, alone or in combination, significantly enhanced the anti-tumor efficacy of oncolytic viral therapy.

  Furthermore, treatment of tumors in mice with two antibody-expressing VACV, GLV-1h444 (anti-EGFR and anti-VEGF) or GLV-1h446 (anti-FAP and anti-VEGF) was surprisingly Superior to the combined treatment with GLV-1h68 combined with sequential administration of avastatin (anti-VEGF) and erbitux (anti-EGFR). The anti-proliferative and angiogenesis-inhibiting effects of virus-expressed antibodies are also evident in tumors beyond the area directly affected by the virus, whereby the scAb is expressed locally while It is demonstrated that it can penetrate. Thus, our results indicate that oncolytic viral therapy using VACV was significantly enhanced by the co-expression of virus-encoded antibodies with anti-proliferative and angiogenesis-inhibiting activities targeting TME. Has been demonstrated. Furthermore, this enhanced treatment effect was achieved by a single dose of replication competent recombinant VACV.

  Thus, a novel recombinant VACV expressing antibodies targeting VEGF, EGFR, and FAP, either alone or in combination, significantly enhanced oncolytic viral therapy in preclinical animal models. The therapeutic efficacy of GLV-1h164, GLV-1h442, and GLV-1h282, each expressing scAb, is significantly better than that of their parental virus GLV-1h68, thereby enabling oncolytic viral therapy. Improved by viral expression of individual antibodies against VEGF, reduced angiogenesis, improved by viral expression of individual antibodies against EGFR, suppressed cell proliferation, or viral expression of individual antibodies against FAP Is shown to be able to reduce angiogenesis and suppress MSC recruitment. Furthermore, this therapeutic efficacy was further enhanced by expressing two antibodies in one VACV strain. The therapeutic efficacy of GLV-1h444 expressing antibodies targeting both VEGF and EGFR is significantly superior to the efficacy of combined treatment with Avastin and erbitux, and GLV in combination with avastatin and erbitux It was superior to the treatment with -1h68.

  High level expression of VEGF, EGFR and FAP in tumors is associated with poor prognosis prediction. Despite promising results in preclinical trials, avastatin and erbitux show only limited clinical efficacy, which is due in part to poor antibody penetration and tumor targeting Low clearance as well as rapid clearance from circulation after systemic administration. Oncolytic VACV not only specifically targets and destroys tumor cells, but also mediates local production of therapeutic proteins in colonized tumors, thereby avoiding the limitations associated with the use of antibody therapy To do. The continuous presence of the antibody was demonstrated in the sera of mice treated with VACV expressing either one or two antibodies. This antibody was detected in higher amounts in the early stage (7 and 21 dpi) after virus injection when the tumor had already started shrinking than in the late stage (35 dpi). Furthermore, anti-FAP (GLAF-5) and anti-EGFR VHHFLAG antibodies were found to be anti-VEGF (GLAF) in the serum of mice treated with GLV-1h282 and GLV-1h442, respectively. -2) It occurred in a higher amount than the antibody. This was consistent with GLV-1h282 and GLV-1h442 having higher virus titers in tumors than GLV-1h164. The viral titer of GLV-1h68 in the tumor was significantly higher than that of GLV-1h164 (P = 0.02), but GLV-1h164 was slightly less in culture than GLV-1h68 It was suggested that the expression of GLAF-2 reduced viral replication in the tumor. Expression of antibodies targeting EGFR or FAP had no negative effect on virus replication in tumors.

  It is well known that cancer progression is due to uncontrolled growth of cancer cells. Treatment with GLV-1h68 suppressed cell growth in the tumor, which was evident in a dramatic reduction in the number of Ki67 + cells in the infected area, consistent with the intrinsic characteristics of VACV infection. . In particular, treatment with GLV-1h442 and GLV-1h444, which express anti-EGFR Nanobodies, significantly reduced the number of Ki67 + cells not only in the infected area, but also in the uninfected area, thereby It is suggested that the anti-EGFR Nanobody secreted from the bacterium propagates to uninfected areas and suppresses the growth of EGFR + cells. Treatment with GLV-1h164 expressing anti-VEGF antibody or GLV-1h282 expressing anti-FAP antibody significantly suppressed the growth in the infected area of the tumor, but the effect was negligible. In the uninfected area, it was not significant. Importantly, treatment with GLV-1h446 expressing both anti-VEGF and anti-FAP significantly suppressed proliferation in both infected and uninfected areas, indicating that two It demonstrates the added inhibitory effect of antibody expression.

  Cancer growth and development also requires a continuous supply of nutrients through blood vessels. A decrease in blood vessel density (BVD) was observed in the infected area but not in the uninfected area of the GLV-1h68 colonized tumor, which may lead to VACV infection itself leading to destruction of the tumor vasculature Is suggested. The expression of anti-VEGFscAb not only enhanced vascular destruction in the infected area, but also resulted in a significant decrease in BVD in the uninfected area. Thus, like anti-EGFR Nanobodies, anti-VEGF scAbs can also propagate from infected areas to uninfected areas. Therapies that target FAP inhibit tumor growth in part through suppression of tumor angiogenesis. Anti-FAP scAb expressed in virus significantly reduced BVD in both the infected and uninfected areas of the FaDu tumor xenograft model and in the infected area of the DU145 tumor xenograft model. Although expression of anti-EGFR Nanobodies did not significantly affect BVD in either the infected or uninfected areas in the DU145 tumor xenograft model, EGFR was reported to promote tumor angiogenesis.

  FAP is one of the markers expressed by cancer-associated fibroblasts (CAF) that support tumor growth. FAP + CAF ablation has been shown to suppress tumor growth. Treatment of mice bearing FaDu tumor xenografts with GLV-1h282 expressing anti-FAP scAb resulted in treatment of FAP + cells in both the infected and uninfected areas of the tumor compared to treatment with GLV-1h68. The number was greatly reduced. Since FaDu tumor cells do not express FAP, the enhanced anti-tumor effect of GLV-1h282 is likely due to an effect on CAF rather than on cancer cells.

  Thus, the oncolytic VACV infection itself greatly reduced the cell growth and vascularity of the colonized tumor. These anti-tumor effects were significantly enhanced by the expression of scAbs against VEGF, EGFR, and FAP, either alone or in combination in recombinant VACV. The effect of co-expression of antibody (s) is comparable or better than treatment with VACV in combination with clinical antibodies, with the benefit of single dose and local intratumoral delivery. It was. This therapeutic effect combined the viral disintegration of infected tumor cells with the antitumor modification of TME. To the best of our knowledge, this is because the virus-expressed antibodies against EGFR and FAP alone enhanced oncolytic virology and combined with anti-VEGF antibodies further improved anti-tumor therapeutic efficacy. This is the first report demonstrating this.

Definitions As used herein, a subject includes any animal for which diagnosis, screening, monitoring or treatment is contemplated. Animals include mammals such as primates and livestock. In some embodiments, the subject is a mammal. In some embodiments, the subject is a mammal selected from a mouse, rabbit, dog or cat. In some embodiments, the subject is a primate. An exemplary primate is a human. A patient refers to a subject, eg, a mammal, primate, human or livestock subject, who is suffering from or has a confirmed disease state or is at risk for a disease state.

  As used herein, the term “antibody” is used in the broadest sense, specifically, as long as they exhibit the desired biological activity, monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, Includes multispecific antibodies (eg, bispecific antibodies), bivalent specific T cell engager (BiTE) antibodies, and antibody fragments (eg, single chain, Nanobodies, etc.). In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a Fab fragment. In some embodiments, the antibody is a single chain antibody. In some embodiments, the antibody is a Nanobody. In some embodiments, the antibody is selected from Fab, Fv, F (ab ') 2, scFV, diabody, and bispecific antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, any antibody embodiment referred to herein is specifically excluded.

  As used herein, “virus” refers to the reality of any large group called a virus. Viruses generally contain a protein coat that surrounds the RNA or DNA core of genetic material, but are capable of propagation and replication only in living cells, not semi-permeable membranes. Viruses for use in the methods provided herein include, but are not limited to, poxvirus, adenovirus, herpes simplex virus, Newcastle disease virus, vesicular stomatitis virus, mumps virus, influenza virus , Measles virus, reovirus, human immunodeficiency virus (HIV), hantavirus, myxoma virus, cytomegalovirus (CMV), lentivirus and any plant or insect virus. In some embodiments, any viral embodiment referred to herein is specifically excluded.

  As used herein, the term “viral vector” is used according to its art-recognized meaning. This refers to a nucleic acid vector construct that contains at least one element of viral origin and can be packaged into a viral vector particle. Viral vector particles may be used for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Viral vectors include, but are not limited to, retroviral vectors, vaccinia vectors, lentiviral vectors, herpes virus vectors (eg, HSV), baculovirus vectors, cytomegalovirus (CMV) vectors, papilloma virus vectors, simian Examples include viral (SV40) vectors, Semliki Forest virus vectors, phage vectors, adenovirus vectors, and adeno-associated virus (AAV) vectors.

  As used herein, “hematological malignancy” refers to tumors of the blood and lymphatic system (eg, Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, malignant immunoproliferative disease, multiple bone marrow Tumors and malignant plasma cell neoplasms, lymphoid leukemia, spinal leukemia, acute or chronic lymphocytic leukemia, monocyte leukemia, other leukemias of certain cell types, leukemias of unspecified cell types, other and unspecified lymphoid System, hematopoietic system and related tissue malignant neoplasms, such as diffuse large cell lymphoma, T cell lymphoma or cutaneous T cell lymphoma).

Recombinant virus and methods of use Disclosed herein, in some embodiments, is a method for treating a solid tumor comprising a recombinant virus capable of infecting cells in the tumor. Or providing the subject with a cell comprising a virus capable of infecting cells in the tumor, wherein the virus expresses two or more of: (i) the tumor microenvironment (TME) Or (ii) a stimulatory or inhibitory protein that targets TME. In some embodiments, the virus is an oncolytic virus. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the virus is a replication competent oncolytic vaccinia virus (VACV). In some embodiments, the method further comprises providing an additional virus to the subject, wherein the virus is: (i) an antibody that targets the tumor microenvironment (TME) or (ii) Expresses a stimulatory or inhibitory protein that targets TME, wherein said at least one additional virus is (i) an antibody that targets the tumor microenvironment (TME) or (ii) a stimulatory or Express one or more of (i) an antibody that targets TME or (ii) a stimulatory or inhibitory protein that targets TME, which is different from that expressed by a virus that expresses the inhibitory protein.

  Accordingly, provided herein are viruses for therapeutic and diagnostic uses, including recombinant vaccinia viruses, which contain a heterologous nucleic acid molecule that contains at least two therapeutic genes. Encode the product (ie, two or more of the following: (i) an antibody that targets the tumor microenvironment or (ii) a stimulatory or inhibitory protein that targets the tumor microenvironment). Such therapeutic gene products can be operably linked to any suitable promoter (eg, vaccinia promoter, eg, vaccinia early promoter, vaccinia middle promoter, vaccinia early / late promoter and vaccinia late promoter).

  In some embodiments, the two or more antibodies that target the tumor microenvironment (TME) are selected from the group consisting of: (i) an antibody that binds to a protein that stimulates angiogenesis and / or angiogenesis. (Iii) an antibody that binds to a receptor tyrosine kinase selected from the group consisting of epidermal growth factor receptor (EGFR), Her2 / C-neu, Her3 and Her4 (Erb family), and (iii) epithelial-mesenchymal An antibody that binds to a protein involved in the development of an interaction. Exemplary proteins that stimulate angiogenesis and / or angiogenesis include paracrine factors (eg, angiogenin, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor-β ( TGF-β) and integrins Proteins involved in the development of epithelial-mesenchymal transition include fibroblast activation protein (FAP), HSP47, collagen 1, collagen 2, vimentin FSP1, DDR2, N-cadherin, Snail, Slug and Twis, OB-cadherin, integrins, syndecan-1, FSP-1, betacatenin, fibronectin, laminin 5, ZEB1, LEF-1, Ets-1, FOXC2 and goosecoid In some embodiments, the two or more antibodies include antibodies specific for VEGF, angiogenin, FGF, TGF-β, or integrins (eg, αVβ3, αVβ5, or α5β1). In some embodiments, the two or more antibodies include antibodies specific for EGFR, Her2 / c-neu, Her3, or Her 4. In some embodiments, the two or more antibodies include: FAP, HSP47, collagen 1, collagen 2, vimentin FSP1, DDR2, N-cadherin, Snail, Slug and Twis, OB-cadherin, integrins, syndecan-1, FSP-1, beta catenin, fibronectin, laminin 5 , ZEB1, LEF-1, ts-1, include antibodies specific to FOXC2 or goosecoid. In some embodiments, any of the embodiments of the antigens listed in this paragraph is specifically excluded.

  In some embodiments, the two or more antibodies are selected from the group consisting of: an antibody that binds to vascular endothelial growth factor (VEGF), an antibody that binds to epidermal growth factor receptor (EGFR), And an antibody that binds to fibroblast activation protein (FAP). In some embodiments, one of the two or more antibodies is an antibody that binds to VEGF. In some embodiments, the antibody that binds to VEGF is G6-31. In some embodiments, the antibody that binds to VEGF is selected from the following: 4G3, ab52917, ab68334, ab46154, A-20, OTI4E3, Ab-3. , SP28. An antibody that can bind to the same epitope as bevacizumab, ranibuzumab, pazopanib, sorafenib, sunitinib, vandetanib, cabozantinib, ponatinib, axitinib and aflibercept, and any of these antibodies. In some embodiments, one of the two or more antibodies is an antibody that binds to EGFR. In some embodiments, the antibody that binds to EGFR is anti-EGFR VHH. In some embodiments, the antibody that binds to EGFR is: cetuximab, matuzumab, panitumumab, nesitumumab, nimotuzumab, trastuzumab, saltumumab, 528, SC-03, DR8.3, DH8.3, L8A4, Y10, ICR62, ABX -Selected from EGF, EMD72000, MM-151, Sym004, mAB806, and antibodies capable of binding to the same epitope as any of these antibodies. In some embodiments, one of the two or more antibodies is an antibody that binds to FAP. In some embodiments, the antibody that binds to FAP is M036. In some embodiments, the antibody that binds to FAP is ab54651, vF19, ESC11, ESC14, F11-24, SS-13, D394, MAb clone 427819, M05, M02, M01, LS-C348807, 2F2. And antibodies that can bind to the same epitope as any of these antibodies. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to EGFR. In some embodiments, the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to FAP. In some embodiments, the two or more antibodies include an antibody that binds to EGFR and an antibody that binds to FAP. In some embodiments, any particular antibody listed in this paragraph or an antibody that binds to the same epitope as a particular antibody listed in this paragraph is specifically excluded.

  In some embodiments, the invention disclosed herein expresses antibodies against VEGF, but consists of the epidermal growth factor receptor (EGFR), Her2 / C-neu, Her3 and Her4 (Erb family) Viruses that do not express antibodies or proteins that bind to a more selected receptor tyrosine kinase and that do not express antibodies or proteins that bind to proteins involved in the development of epithelial-mesenchymal interactions are not included.

  Exemplary oncolytic viruses include vaccinia virus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), retrovirus, reovirus, measles virus, simbis virus, influenza virus, herpes simplex virus (HSV). ), Vaccinia virus and adenovirus.

  In some embodiments, the viruses disclosed herein are attenuated. Techniques for attenuating viruses are known in the art.

  Exemplary viruses provided herein include a recombinant vaccinia virus comprising a modified hemagglutinin (HA) gene, a thymidine kinase (TK) gene, and an F14.5L gene, one of which One or more includes the insertion of a heterologous non-coding nucleic acid molecule into the HA, TK, or F14.5L locus. In such viruses, functional HA, TK and F14.5L polypeptides are not expressed.

  Exemplary viruses provided herein for therapeutic and diagnostic use also include Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD -J and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC16MO, LIVP, WR 65-16, Connaught, New York City Department of Health. LIVP vaccinia viruses described herein for use in the methods described herein include GLV-1h22, GLV-1h68, GLV-1i69, GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h75, GLV-1h81, GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89, GLV-1h90, GLV- 1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h104, GLV-1h105, GLV-1h106. Exemplary LIVP vaccinia viruses provided herein for use in the methods described herein include GLV-1h107, GLV-1h108, and GLV-1h109. In some embodiments, the virus for use in the described methods is selected from GLV-1h107, GLV-1h108 and GLV-1h109.

  In some embodiments, the claimed virus is selected from GLV-1h444 and GLV-1h446.

  In some embodiments, the viruses provided herein for therapeutic and diagnostic use include recombinant vaccinia comprising a heterologous nucleic acid molecule encoding a detectable protein or a protein capable of inducing a detectable signal. Virus. Examples of such proteins include luciferases such as click beetle luciferase, Renilla luciferase, or firefly luciferase, fluorescent proteins such as GFP or RFP, or proteins that can bind to contrast agents, chromophores, or compounds that can be detected Or a ligand, such as a transferrin receptor or ferritin. Provided herein is the claimed recombinant Lister strain vaccinia virus, which encodes click beetle luciferase (CBG99) and RFP (eg, GLV-1h84).

  Provided herein are viruses for therapeutic and diagnostic applications, including two or more diagnostic or therapeutic gene products in which the gene product is linked to a picornavirus 2A element. Heterologous nucleic acid molecules encoding are included. In one example described herein, a recombinant vaccinia virus comprises a heterologous nucleic acid molecule that encodes CBG99 linked by a picornavirus 2A element to a second heterologous nucleic acid molecule. This second heterologous nucleic acid molecule then encodes RFP (eg, GLV-1h84).

  In some embodiments, described herein are recombinant vaccinia viruses for therapeutic and diagnostic use, including replacement of the A34R gene with an A34R gene from another vaccinia virus strain. Provided herein is the claimed Lister strain vaccinia virus, wherein the A34R gene is replaced with an A34R gene from a vaccinia IHD-J strain (eg, GLV-1 i69). Such substitution increases the virus (EEV) form of the extracellular envelope of vaccinia virus and increases the resistance of the virus to neutralizing antibodies.

  Described herein are recombinant vaccinia viruses for therapeutic and diagnostic uses, including deletion of the A35R gene.

  Described herein are therapeutics that can be further modified by the addition of one or more additional heterologous nucleic acid molecules encoding a therapeutic protein, a detectable protein, or a protein that can induce a detectable signal. And a recombinant vaccinia virus for diagnostic use. Examples of such proteins include luciferases such as click beetle luciferase, Renilla luciferase, or firefly luciferase, fluorescent proteins such as GFP or RFP, or proteins that can bind to contrast agents, chromophores, or compounds that can be detected or There are ligands such as transferrin receptor or ferritin. In some embodiments, the diagnostic protein comprises a luciferase, a fluorescent protein, an iron storage molecule, an iron transporter, an iron receptor, a protein that binds to an iron receptor or a contrast agent, a chromophore, or a detectable compound or detectable ligand It is chosen from among them.

  Also included in such methods are therapeutic gene products such as cytokines, chemokines, immunomodulatory molecules, single chain antibodies, antisense RNA, siRNA, prodrug converting enzymes, biological toxins, antitumor oligopeptides Insertion of a heterologous nucleic acid molecule encoding an anti-cancer polypeptide antibiotic, an angiogenesis inhibitor, or tissue factor. Exemplary antigens include tumor specific antigens, tumor associated antigens, tissue specific antigens, bacterial antigens, viral antigens, yeast antigens, fungal antigens, protozoal antigens, parasite antigens and mitogens. . One or more additional heterologous nucleic acid molecules encoding a therapeutic protein, a detectable protein, or a protein that can induce a detectable signal can be operably linked to a promoter, such as a vaccinia virus promoter. In some embodiments, the therapeutic protein is a cytokine, chemokine, immunomodulatory molecule, antigen, single chain antibody, antisense RNA, prodrug converting enzyme, siRNA, angiogenesis inhibitor, biological toxin, antitumor oligo Selected from among peptides, antimitotic proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics and tissue factor.

  Provided herein are host cells comprising the claimed recombinant virus. An exemplary host cell is a tumor cell containing a recombinant virus as claimed. In some embodiments, the host cell is a mammalian cell line in culture. In some embodiments, the host cell is a human cell line in culture.

  Also disclosed herein are mammalian organisms that contain or are infected by a recombinant virus as claimed. In some embodiments, the mammalian organism is a mouse, rat, rabbit, or monkey.

  Provided herein is a pharmaceutical composition comprising the claimed recombinant virus and a pharmaceutically acceptable carrier. The composition comprises the virus in an amount or concentration appropriate for the intended use, eg, treatment, diagnostic method or both, and route of administration. Provided herein are such pharmaceutical compositions formulated for local or systemic administration. Provided herein is a pharmaceutical composition comprising two or more viruses. Provided herein is a pharmaceutical composition formulated for administration as a vaccine, such as smallpox vaccine.

  Provided herein is a pharmaceutical composition for use to treat a tumor, cancer or metastasis in a subject, such as a human or animal subject. Administration of the pharmaceutical composition stops or delays tumor growth, reduces tumor volume, or causes the tumor to be removed from the subject.

  The methods and pharmaceutical compositions disclosed herein may be used to treat any solid tumor or hematological malignancy. Tumors that can be treated by the methods disclosed herein include, but are not limited to, bladder tumors, breast tumors, prostate tumors, carcinomas, basal cell cancers, bile duct cancers, bladder cancers, bone cancers, brain tumors, CNS Cancer, glioma, cervical cancer, choriocarcinoma, colon cancer and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, Kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, retinoblastoma, striated muscle Tumor, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary cancer, eg lymphosarcoma, osteosarcoma, breast tumor, mastocytoma , Brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland Tumor, bronchiole adenocarcinoma, small cell lung cancer, non-small cell lung cancer, fibroma, myxochondroma, lung sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing sarcoma, Wilms tumor, Burkitt lymphoma, small Glioma, neuroblastoma, giant cell tumor, oral neoplasm, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, reproductive squamous cell carcinoma, transplantable genital tumor, testicular tumor, seminoma, Sertoli cell tumor , Hemangiocytoma, histiocytoma, green tumor, granulocyte sarcoma, corneal papilloma, squamous cell carcinoma of the cornea, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenal carcinoma, oral papilloma Disease, hemangioendothelioma, bladder adenoma, vesicular lymphoma, intestinal lymphosarcoma, fibrosarcoma and lung squamous cell carcinoma, leukemia, hemangiovascular carcinoma, ocular tumor formation, foreskin fibrosarcoma, ulcerative squamous cell carcinoma, foreskin carcinoma Connective tissue tumor formation, mastocytoma, liver cancer, lymph , Pulmonary adenomatosis, pulmonary sarcoma, Rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B cell lymphoma, lymphocytosis, retinoblastoma, liver tumor formation, lymphosarcoma, plasmacytoid leukemia (Fish), dry toy lymphadenitis, lung cancer, insulinoma, lymphoma, sarcoma, neuroma, islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma. In some embodiments, the tumor is selected from: glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumor, and melanoma.

  The pharmaceutical compositions provided herein can be systemically, intravenously, intraarterially, intratumorally, endoscopically, focal, intramuscularly, intradermally, intraperitoneally, Intra-alveolar, intra-articular, intra-pleural, percutaneous, subcutaneous, buccal, parenteral, intranasal, intratracheal, intracranial, intraprostatic, intravitreal Alternatively, it may be administered topically, ocularly, vaginally or rectally.

  The pharmaceutical compositions provided herein may be administered together with antiviral agents such as cidofovir, alkoxyalkyl esters of cidofovir, Gleevec, ganciclovir, acyclovir and ST-26. It is not limited.

  Provided herein is a claimed combination comprising a pharmaceutical composition provided herein and an anticancer agent. Exemplary anticancer agents for use in the combinations provided herein include, but are not limited to, cytokines, chemokines, growth factors, photosensitizers, toxins, anticancer antibiotics, chemotherapeutic compounds, radioactive Nuclide, angiogenesis inhibitor, signal transduction regulator, antimetabolite, anticancer vaccine, anticancer oligopeptide, antimitotic protein, antimitotic oligopeptide, anticancer antibody, anticancer antibiotic, immunotherapy Agents, hyperthermia or thermotherapy, bacteria, radiation therapy or combinations thereof. Exemplary chemotherapeutic compounds for use in the combinations provided herein include, but are not limited to, alkylating agents, such as, among other chemotherapeutic compounds provided herein. And platinum coordination complexes. Exemplary platinum coordination complexes include, but are not limited to, cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S.

  Provided herein are claimed viruses and anticancer agents, such as cytokines, chemokines, growth factors, photosensitizers, toxins, anticancer antibiotics, chemotherapeutic compounds, radionuclides, angiogenesis inhibitors, Signal transduction regulator, antimetabolite, anticancer vaccine, anticancer oligopeptide, antimitotic protein, antimitotic oligopeptide, anticancer antibody, anticancer antibiotic, immunotherapeutic agent, hyperthermia or thermotherapy, Or a combination of bacteria. Provided herein are combinations of claimed viruses and anti-cancer agents such as cisplatin, carboplatin, gemcitabine, irinotecan, anti-EGFR antibody and anti-VEGF antibody.

  Provided herein are combinations as claimed where the compound and virus are formulated separately in the two compositions. Provided herein are claimed combinations where the compound and virus are formulated as a single composition.

  Provided herein are the viruses claimed herein for use in the treatment of tumors, cancers or metastases. Also provided herein is the use of the virus claimed herein for the preparation of a pharmaceutical composition for the treatment of a tumor, cancer or metastasis.

  Provided herein is a kit comprising a pharmaceutical composition or combination claimed herein, and optionally instructions for its administration for the treatment of cancer.

  Provided herein are vaccines, such as smallpox vaccines, including the recombinant vaccinia virus claimed herein. Further provided herein is a recombinant vaccinia virus as claimed herein for administration as a vaccine, such as smallpox vaccine, to a subject for generating an immune response.

  In some embodiments, disclosed herein is a method of sensitizing a tumor to a subsequent treatment regime. The sensitization part of the technique according to some embodiments may be performed using any of the approaches described herein.

  In some embodiments, the subsequent treatment modality is selected from the group consisting of: radiation therapy, chemotherapy, immunotherapy, phototherapy, or a combination thereof. In some embodiments, sensitizing the tumor includes irradiating the subject. In some embodiments, the irradiation is ionizing radiation. In one embodiment, this sensitization is achieved with local tumor irradiation, eg, high dose subdivision radiation therapy (HDHRT).

  Ionizing radiation has a significant ability to modify the tumor microenvironment and facilitate immune-mediated tumor rejection. Specifically, radiation can induce abnormal tumor blood vessel remodeling and upregulation of vascular cell adhesion molecules (eg, VCAM-1) and chemokine secretion (eg, CXCL16), thereby causing tumor Effective T cell infiltration into the cell occurs. Other important effects of radiation include upregulation of MHC class I molecules, NKG2D ligands, and Fas / CD95, which enhances T cell binding and killing to cancer cells. However, despite these significant immunogenic effects, radiation itself is insufficient to induce a persistent and strong anti-tumor immune response to effect tumor eradication.

  Radiation therapy includes, but is not limited to, photodynamic therapy, radionuclide, radioimmunotherapy and proton beam treatment.

  In some embodiments, the subsequent treatment modality includes administration of a chemotherapeutic compound. Chemotherapeutic compounds include, but are not limited to, platinum; platinum analogs (eg, platinum coordination complexes) such as cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295 and 254-S; anthracenedione Vinblastine; alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piperosulphan; and aziridines such as benzodopa, carbocon, metresorpa and uredopa; ethyleneimine and methylammelamine, eg Examples include altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine nitrogen mustard such as chlorambucil ( chiorambucil), chlornafazine, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitine, phenesterin, prednisomine, trophosphamide, uracil mustard; Antibiotics such as acracinomycin, actinomycin, anthramycin, azaserine, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, Detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin Epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogaramycin, olivomycins, pepromycin, potomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozosine, tubercidin, ubenimex, dinostatin, zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterine, methotrexate, pteropterin, trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiapurine, thioguanine; pyrimidine Analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuri , Doxyfluridine, enocitabine, floxuridine; androgens, such as carsterone, drmostanolone propionate, epithiostanol, mepithiostan, test lactone; anti-adrenals, such as aminoglutethimide, mitotane, Folic acid supplements such as floric acid; acegraton; aldophosphamide glycoside; aminolevulinic acid; amsacrine; vesturacil; bisantrene; edatotraxate; defofamine; demecoltin; Eliptinium acetate; etoglucide; gallium nitrate; substituted urea; hydroxyurea; lentinan; lonidamine; mitguazone; mitoxantrone; Nitracline; Pentostatin; Phenamestat; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Anticancer polysaccharide; Polysaccharide-K; Razoxane; Sizophyllan; Spirogermanium; Tenuazonic acid; Triadicon; '-Trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitoblonitol; mitractol; Mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; Stin; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; XELODA; Ibandronate; CPT11; Topoisomerase inhibitor RFS 2000; TARCEVA); sunitinib malic acid (SUTENT); pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormonal effects on tumors, such as antiestrogens such as tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4- Hydroxytamoxifen, trioxyphene, keoxifene, LY11018, onapristone, and toremifene (FARESTON); adrenocortical inhibitors; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any of the above A pharmaceutically acceptable salt, acid, or derivative. Such chemotherapeutic compounds that can be used herein include compounds in which the use of the compound in general systemic chemotherapy is inhibited by toxicity.

  In some embodiments, the subsequent treatment modality is selected from: local tumor irradiation, cytokine injection, antibody injection, and injection of stem cells secreting cytokines and / or chemokines.

Treatment regimen management The effective dose of each of the treatment modalities disclosed herein, including the recombinant viruses disclosed herein, used in the combination therapy of the invention is the specific treatment, compound used Or the pharmaceutical composition, the mode of administration, the disease being treated, and the severity of the disease being treated. Accordingly, the dosage regimen for treatment according to the present invention is selected according to various factors including the route of administration and the renal and liver function of the patient. A physician, clinician or veterinarian of ordinary skill in the art will determine the effective amount of a single active ingredient necessary to prevent, combat or stop disease progression. Can be easily determined and prescribed. Optimal accuracy in achieving concentrations of the active ingredient within the range that yields efficacy without causing toxicity requires a regimen based on the kinetics of the active ingredient's availability to the target site.

  Methods of preparing pharmaceutical compositions, including related treatments disclosed herein, are known in the art and are known from the art, and are known standard references such as Remington's Pharmaceutical Sciences. , Mack Publishing Company, Easton, Pa. , 18th edition (1990).

Example 1-Materials and Methods Cell lines African green monkey kidney fibroblast CV-1, human lung cancer A549, hypopharyngeal cancer cell line FaDu, and human prostate cancer cell line DU145 were obtained from the American Type Culture Collection (American Type Culture Collection). ). CV-1 cells were cultured in DMEM; A549 cells were cultured in RPMI 1640; FaDu and DU145 were cultured in Eagle's minimum essential medium. All media was supplemented with 10% fetal calf serum (Cellgro).

Plasmid construction and antibody expression The parental triple mutant VACV GLV-1h68 was constructed as previously described (Zhang et al., Cancer Res. 67: 10038-10046). In summary, it encodes Renilla luciferase-Aequorea GFP fusion protein (RUC-GFP), β-galactosidase, and β-glucuronidase, integrated into the F14.5L, J2R and A56R loci, respectively, of the LIVP virus genome. Contains two foreign gene expression cassettes. The sequence of GLAF-5 was designed as previously described (Frentzen et al., Proc. Natl. Acad. Sci. USA 106: 12915-12920) and synthesized by GENEART-Invitrogen. A DYKDDDDK tag (gac tac aag gat gac gac gac aag) was added to the C-terminal coding region of anti-EGFR VHH to obtain anti-EGFR VHHFLAG. The DNA fragment was cloned into the Sal I and Pac I sites of plasmid pCR-TK-P_SEL to obtain plasmids pCR-TK-P_SEL (GLAF-5) and pCR-TK-P_SEL (anti-EGFRVHHFLAG), which Used for homologous recombination into the J2R locus in GLV-1h68 via double reciprocal crossing, thereby obtaining GLV-1h282 and GLV-1h442, respectively. GLV-1h446 and GLV-1h444 were similarly generated using the same plasmid and GLV-1h164 as the parental virus. The sequence of the recombinant VACV was confirmed. All VACVs were grown in CV-1 cells and purified via a sucrose gradient by standard protocols.

  For detection of antibody expression, cell samples were harvested 24 hours after infection with each VACV strain at 1 MOI and separated by 12% SDS-PAGE. The following antibodies were used: anti-DDDDK antibody (Abcam) for detection of FLAG tag, custom-made antibody G6 for detection of scAb, and anti-A27 antibody (GenScript Corporation for detection of VACV membrane protein) ).

  Antibody concentrations in mouse serum are standard using commercially available recombinant human EGFR (Abnova), FAP (R & D system), and VEGF (Sigma) as previously described (Frentzen et al., Cited above). Determined by ELISA assay.

Virus replication assay Cells were infected with VACV at a MOI of 0.01 and samples were collected in triplicate at each time point after infection.

Animal Testing Male nude mice (Hsd: athymic nude-Foxn1nu mice), 5-6 weeks old, were purchased from Harlan (Livermore, CA) and tested in Animal Biolabs (San Diego Science Center, San Diego, CA). Handled and maintained under a protocol approved by the Committee (Institutional Animal Care and Use Committee). 1 × 10 6 FaDu, 5 × 10 6 A549, or 1 × 10 7 DU145 cells were implanted subcutaneously into the right hind limb of mice. Tumor volume was measured weekly in three dimensions using a digital caliper and calculated as ((length × width × (height−5)) / 2) and body weight was measured weekly. The net body weight of each animal was calculated by subtracting the total body weight at each time point from the weight of the tumor (taken as 1 g tumor density per cc). The percent change in net body weight is expressed as a percentage of the difference between the net weight of each animal at a particular time point and its net body weight just before treatment divided by the net weight just before treatment. Was. When tumor volume reached approximately 450 mm 3, 100 μl of PBS containing a single dose of 2 × 10 6 pfu / mouse (unless otherwise specified) of each VACV strain was administered by retroorbital injection. 100 microliters of PBS was injected as a negative control.

  For treatment with therapeutic antibodies, mice were given intraperitoneal (i) Avastin (5 mg / kg) and / or Erbitux (3 mg / kg) twice a week for 5 weeks starting at 10 dpi. P.) Injection.

  Tumors and organs were excised and homogenized using MagNA Lyser (Roche Diagnostics). Virus titer was determined by standard plaque assay in CV-1 cells.

Histological analysis FaDu tumors were excised and embedded in paraffin, followed by a standard dehydration process. Tumor samples were cut into 5 μm sections and stained with hematoxylin and eosin (H & E). The sections were dewaxed and antigen activation was performed in sodium citrate buffer. The following antibodies were used: anti-FAP (Abcam) and anti-A27L (GenScript Corporation). Biotinylated secondary antibody (goat anti-rabbit; Jackson ImmunoResearch Laboratories) was used, and detection was performed using Vectorstein Elite ABC reagent and Vector ImPact DAB peroxidase substrate (Vector Laboratories).

  DU145 tumors were excised at 36 dpi followed by paraformaldehyde fixation and then excised into 100 μm sections. Blood vessel and cell proliferation were detected using anti-CD31 antibody (BD Pharmingen) and anti-Ki67 antibody (BD Pharmingen), respectively.

  Tumor sections were examined with an MZ16FA fluorescent stereomicroscope (Leica) equipped with a digital electrified coupling device camera (Leica). Digital images (1,300 to 1,030 pixel images) were processed using Adobe Photoshop 7.0 software.

Statistical analysis Statistically significant differences between groups of animals were analyzed using unpaired two-tailed Student's t-test (Excel 2003; Microsoft, Redmond, WA) or one-way analysis of variance (ANOVA) in STATISTICS. A p value <0.05 was considered significant.

Example 2-Construction of recombinant VACV encoding individual antibodies targeting EGFR and FAP VACV (GLV-lh108 (cited 29) and GLV-lh164 (Buckel et al., Int. J. Cancer 133: 2989-2999) ) -Expressed anti-VEGF scAb (GLAF-1 or GLAF-2) significantly reduced tumor growth in several human tumor xenograft models, with an inhibitory effect on vascular distribution in TME. It has been shown previously that it has exhibited an “abastine-like mode of action.” EGFR and FAP are other important factors in TME that are involved in the regulation of tumor development and development. To evaluate the effects of antibodies targeting EGFR and FAP on therapeutic effects, two new The recombinant VACV is an anti-EGFR Nanobody (anti-EGFRVHHFLAG) expression cassette or an anti-FAP scAb (GLAF-5) expression cassette, both under the control of the VACV synthetic early / late (PSEL) promoter, and the J2R of GLV-1h68 At the locus, it was constructed by replacing the lacZ expression cassette, resulting in GLV-1h442 and GLV-1h282, respectively (FIGS. 1a, 1b).

Example 3 Viral Expressed Antibodies Targeting VEGF, EGFR, and FAP Significantly Improve Viral Therapy Anti-Tumor Treatment with VACV Strains Encoding Anti-VEGF, Anti-FAP scAb, or Anti-EGFR Nanobodies The effect was examined in mice. VACV strain or PBS (phosphate-interfering saline) was injected into the retro-orbital at a single dose of 2 × 10 6 plaque forming units (PFU) per mouse in mice bearing various human tumor xenografts. Injected. Avastin or erbitux was administered to mice twice a week by intraperitoneal (ip) injection for 5 weeks starting 10 days after virus injection (arrows in FIGS. 2a-2c) As shown). In the A549 xenograft model (n ≧ 7) (FIGS. 2a, b), PBS treated tumors showed continuous growth until mice had to be sacrificed with an excessive tumor burden. A typical three-stage growth pattern of tumors was observed in mice treated with GLV-h168 (Zhang et al., Cited above). Tumor volume exceeded the PBS-treated group at the start, followed by significant tumor growth cessation, followed by continuous tumor shrinkage. Mice treated with Avastin alone showed a decrease in tumor volume compared to PBS, but tumor growth continued (Figure 2a). Treatment with GLV-1h68 in combination with Avastin resulted in improved efficacy over either treatment alone, but the therapeutic effect of GLV-1h164 expressing anti-VEGF scAb was shown to be GLV-1h68 in combination with Avastin. It was almost the same as the treatment effect. Treatment with GLV-1h68 in combination with erbitux resulted in smaller tumors than treatment with GLV-1h68 alone during the erbitux administration period, but tumor volume was reduced after treatment with erbitux was discontinued. Rebounded temporarily (Fig. 2b). In contrast, tumor growth in mice treated with GLV-1h442 expressing anti-EGFR Nanobodies was consistently slower than in mice treated with GLV-1h68, and no tumor volume rebound was observed. It was. Furthermore, treatment of mice with GLV-1h282 expressing anti-FAP scAb also showed significantly smaller tumor volume than treatment with GLV-1h68 (FIG. 2c). Thus, an enhanced therapeutic effect on A549 tumor growth was observed in treatment of mice with GLV-1h164, GLV-1h442, or GLV-1h282, each expressing a therapeutic antibody, compared to GLV-1h68 . Furthermore, this effect was superior to treatment with therapeutic antibody alone, and was comparable to or better than treatment treatment with therapeutic antibody and GLV-1h68. Similar therapeutic effects were also observed in mice bearing DU145 tumor xenografts (n = 5) (FIG. 2d).

Example 4-Effect of intra-tumor expressed antibodies targeting VEGF, EGFR, and FAP against TME The effect of virus-expressed anti-VEGF scAb on tumor blood vessels was measured 36 days after VACV injection (dpi) 36 days The DU145 tumors excised in the above were evaluated. Tumor sections were immunohistochemically (IHC) stained to assess vascular density (BVD) determined by counting CD31 + vessels in the tumor sections. VACV infection was shown by the effect of virally expressed GFP (FIG. 3a). VACV colonization resulted in a dramatic reduction in BVD in the infected area of the tumor compared to both the PBS-treated tumor and the uninfected area of the same tumor (FIG. 3b). This was true for both GLV-1h68 treated tumors and GLV-1h164 treated tumors. However, BVD in uninfected areas of GLV-1h68 treated tumors was not significantly different from BVD in PBS treated tumors. In contrast, treatment with GLV-1h164 significantly reduced BVD in the infected and uninfected areas of the tumor compared to GLV-1h68 treated tumors. Thus, VACV infection alone reduced BVD in the tumor by GLV-1h68, but the effect was confined to the site of infection, while the combination of VACV infection and expression of anti-VEGF scAb by GLV-1h164 was In addition to further reducing BVD in the infected area, the effect on the uninfected area was also expanded.

  It is well known that EGFR overexpression results in uncontrolled cell growth. IHC staining with anti-Ki67 was performed to evaluate whether the virus-expressed anti-EGFR Nanobody suppressed cell proliferation in the tumor. As expected, colonization of tumors with either GLV-1h68 or GLV-1h442 expressing anti-EGFR Nanobodies greatly reduced the number of Ki67 + cells in the infected area compared to PBS-treated tumors ( FIG. 3c). Interestingly, the number of Ki67 + cells in the uninfected area of the GLV-1h442 treated tumor was also significantly reduced compared to the uninfected area of the GLV-1h68 treated tumor (FIG. 3d). This suggested that anti-EGFR Nanobodies secreted from cells infected with GLV-1h442 also act on uninfected cells and reduce their proliferation.

  FAP is a mesenchymal stem cell (MSC) marker that is involved in angiogenesis. The effect of anti-FAP scAb expressed in the tumor was assessed by counting FAP + and CD31 + cells in FaDu tumors that express high levels of FAP. FaDu tumors did not respond to treatment with GLV-1h68, but the growth of FaDu tumors was significantly inhibited by GLV-1h282 expressing anti-FAP scAb (FIG. 4). In GLV-1h68 colony forming tumors, the number of CD31 + and FAP + cells was greatly reduced in the infected area, but no effect was observed in the uninfected area compared to the PBS treated tumor (FIG. 3e, f). In contrast, the number of CD31 + and FAP + cells in GLV-1h282 treated tumors was significantly reduced in both infected and uninfected areas compared to GLV-1h68 treated tumors.

Example 5-Construction of additional novel recombinant VACV expressing two antibodies targeting VEGF and EGFR or VEGF and FAP Based on the positive effect of treatment with VACV expressing a single antibody, VEGF and EGFR or An additional novel recombinant VACV expressing two antibodies targeting VEGF and FAP was constructed. An expression cassette for anti-EGFR Nanobody (anti-EGFRVHHFLAG) or anti-FAP scAb (GLAF-5) is inserted into the J2R locus of GLV-1h164, which also contains an anti-VEGF (GLAF-2) expression cassette, and human norepinephrine transport The body (hNET) expression cassette was replaced, thereby obtaining GLV-1h444 and GLV-1h446, respectively (FIG. 1b). In order to verify the expression of each antibody from the new recombinant VACV, A549 cells were infected with the new VACV strain and the cell lysate was analyzed by Western blot. This result indicated that each antibody was expressed from each virus as intended (FIG. 5).

Example 6 Two Virus Expressed Antibodies have No Negative Effect on Viral Replication Effectiveness The virus replication assay was performed on A549 cells with a multiplicity of infection (MOI) of 0.01 (Figure 6). Recombinant VACV expressing a single antibody or two antibodies showed significantly higher replication efficiency than GLV-1h68 at 24 hours post-infection (hpi). However, there was no significant difference in replication efficiency at 48 hpi or 72 hpi. Similar results were obtained with DU145 cells. Therefore, expression of the two antibodies with VACV did not show a negative effect on their overall replication efficiency in cell culture.

Example 7-Two antibodies of viral expression, targeting TME, further improve viral therapy were expressed from the same VACV, prompted by results from recombinant VACV expressing a single antibody targeting TME An evaluation was made considering whether the two antibodies would further improve viral therapy. A single dose of each VACV strain was injected into mice bearing A549 tumor xenografts (FIGS. 7a, b). Control animals were treated with PBS or a combination of Avastin and erbitux. Treatment with Avastin and Erbitux twice a week for 5 weeks starting at 10 dpi delayed tumor growth during the duration of antibody treatment, but tumor growth reappeared after cessation of antibody treatment. Surprisingly, it was shown that tumors in mice treated with GLV-1h444 or GLV-1h446 expressing the two antibodies grew minimally during the experimental period. In contrast, tumors in mice treated with VACV expressing a single antibody initially grow as fast as tumors treated with PBS, or grow very slightly later, followed by tumor growth Significantly slowed, and then the tumor shrinked. Overall, the tumor volume in mice treated with GLV-1h444 or GLV-1h446 expressing the two antibodies was less than that in mice treated with VACV expressing a single antibody. The tumor growth curve of individual mice bearing A549 tumor is shown in FIG. A similar pattern of tumor growth was obtained with DU145 tumors (FIG. 7c).

Example 8-Virus-expressed antibodies are detectable in the serum of treated mice After confirmation of antibody expression in virus-infected cells in culture, the presence of antibodies in tumor-bearing mice was also examined. Blood samples were collected from the retro-orbital at 7, 21, and 35 dpi from the same mouse. All samples were tested for the presence of anti-FAP scAbs using FAP pre-coated plates, for the presence of anti-EGFR Nanobodies using EGFR pre-coated plates, and for the presence of anti-VEGF scAbs using VEGF pre-coated plates. All antibodies were detectable at all three time points (FIGS. 7d-f). GLAF-2 expression was treated with GLV-1h444 and GLV-1h446 (2 antibodies) at 7 and 21 dpi, consistent with a low viral titer in the tumor at 14 dpi in the GLV-1h164 treated group It was lower in mice treated with GLV-1h164 (single antibody) than in mice (FIG. 9). Nevertheless, in all cases, early stage (day 7 and 21) antibody expression was consistent with low tumor volume, and late stage (35 days) led to tumor shrinkage.

Example 9-Two antibodies of viral expression do not alter virus distribution and toxicity in mice Evaluating potential adverse effects of administration of antibody-expressing VACV in mice, changes in net body weight during treatment Evaluated by. In both A549 and DU145 tumor xenograft models, no significant change in net mean body weight was observed in either the treatment group or the subject group (FIGS. 10a-c). We also assessed the biodistribution of viruses in various organs and tumors at 14 dpi in A549 tumor-bearing mice as determined by standard virus plaque assay (FIG. 9). Significant virus titers were detected in all treated tumors, and no titer or minimum titer was detected in other organs. Viral titers in tumors treated with GLV-1h282 expressing anti-FAP scAb and GLV-1h442 expressing anti-EGFR Nanobodies were higher than in tumors treated with GLV-1h68, but statistically On the other hand, the viral titer of GLV-1h164 expressing anti-VEGF scAb in tumors was significantly lower than other virus strains (GLV-1h164 vs. GLV-1h68, P = 0.02). , And pair GLV-1h446, P = 0.04).

Example 10-Effect of two tumor-expressed antibodies targeting VEGF and EGFR or VEGF and FAP on TME The effect of two virally expressed antibodies on TME was investigated using anti-Ki67 and anti-CD31 antibodies. The tumor sections obtained at 36 dpi were assessed by examining cell proliferation and BVD in DU145 tumor xenografts by staining. VACV infection was confirmed by GFP fluorescence. A representative image of IHC staining of proliferating Ki67 + cells is shown in FIG. 11a. In the infected area of the tumor, Ki67 + cells were significantly reduced after treatment with any of the VACV strains containing GLV-1h68 (FIG. 11c). In uninfected areas of VACV treated tumors, Ki67 + cells are significant only after treatment with a single anti-EGFR Nanobody expressing VACV, GLV-1h442, and two antibody expressing VACV, GLV-1h444 and GLV-1h446 (FIG. 11b).

  Images of DU145 tumor sections stained with anti-CD31 antibody showed that the infected area of the tumor significantly reduced BVD compared to the uninfected area, regardless of the VACV used for treatment. (FIGS. 12a-c). Compared to GLV-1h68, a significant further decrease in BVD was observed with VACV expressing the anti-VEGF antibody (GLAF-2), either alone or in combination with other antibodies. As expected, BVD in tumors treated with GLV-1h442 that expressed anti-EGFR but not anti-VEGF was not significantly different from BVD in tumors treated with GLV-1h68, but GLV-1h444. And the infected area treated with GLV-1h446 showed a further reduction in BVD.

  All publications cited herein are incorporated by reference.

  The disclosure exemplarily described herein may be suitably implemented in the absence of any element (s), restriction (s) that are not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read in a comprehensive and non-limiting manner. Further, the terms and expressions used herein are used as terms of description, not as terms of limitation, and any use of such terms and expressions will be shown and described in the future. It is not intended to exclude the equivalents, or portions thereof, although it will be recognized that various modifications are possible within the scope of the claimed disclosure.

Other embodiments are within the scope of the following claims.

Claims (58)

  A method for treating a solid tumor in a subject comprising administering to said subject a recombinant virus capable of infecting cells in said tumor, or a cell comprising a virus capable of infecting cells in said tumor. The virus is: (i) two or more antibodies that target the tumor microenvironment (TME); (ii) two or more stimulatory or inhibitory proteins that target TME, or (iii) target TME Expressing said at least one antibody and at least one protein that stimulates or inhibits TME.   The method of claim 1, wherein the virus is an oncolytic virus.   The method of claim 2, wherein the oncolytic virus is a vaccinia virus.   4. The method of claim 3, wherein the virus is replication competent oncolytic vaccinia virus (VACV).   5. The method according to any of claims 1-4, wherein the virus expresses two or more antibodies that target TME.   The two or more antibodies are: (a) an antibody that binds to a protein that stimulates angiogenesis and / or angiogenesis, (b) epidermal growth factor receptor (EGFR), Her2 / C-neu, Her3 and Her4. 6. The antibody of claim 5 selected from the group consisting of an antibody that binds to a receptor tyrosine kinase selected from the group consisting of: and (c) an antibody that binds to a protein involved in the development of epithelial-mesenchymal interaction. Method.   The two or more antibodies are: antibodies that bind to vascular endothelial growth factor (VEGF), antibodies that bind to epidermal growth factor receptor (EGFR), and antibodies that bind to fibroblast activation protein (FAP) The method of claim 6, wherein the method is selected from the group consisting of:   8. The method of claim 7, wherein the virus expresses an antibody that binds to VEGF, an antibody that binds to EGFR, and an antibody that binds to FAP.   8. The method of claim 7, wherein one of the two or more antibodies is an antibody that binds to VEGF.   9. The method of claim 8, wherein the antibody that binds to VEGF is G6-31.   8. The method of claim 7, wherein one of the two or more antibodies is an antibody that binds to EGFR.   12. The method of claim 11, wherein the antibody that binds to EGFR is anti-EGFR VHH.   8. The method of claim 7, wherein one of the two or more antibodies is an antibody that binds to FAP.   14. The method of claim 13, wherein the antibody that binds to FAP is M036.   8. The method of claim 7, wherein the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to EGFR.   8. The method of claim 7, wherein the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to FAP.   8. The method of claim 7, wherein the two or more antibodies include an antibody that binds to EGFR and an antibody that binds to FAP.   The method of claim 4, wherein the VACV is selected from GLV-1h444 and GLV-1h446.   19. The method according to any of claims 1-18, further comprising administering an additional cancer treatment.   20. The method of claim 19, wherein the additional cancer treatment is selected from: radiotherapy, chemotherapy, immunotherapy, phototherapy, and combinations thereof.   21. Any of the claims 1-20, wherein the tumor is selected from the following: glioblastoma, breast cancer, lung cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, central nervous system tumor, and melanoma. The method described in 1.   24. The method of any of claims 1-21, wherein the virus is delivered intravenously to the subject.   23. The method of claim 22, wherein the virus is delivered intravenously directly to the tumor or is delivered to the subject in the area of the tumor.   And further comprising providing the subject with at least one additional virus capable of infecting cells within the tumor, wherein the at least one additional virus targets: (i) an antibody that targets the tumor microenvironment (TME) Or (ii) express one or more of the stimulatory or inhibitory proteins targeting TME and expressed by one or more: (i) an antibody targeting TME or (ii) at least one additional virus 2. The method of claim 1, wherein the TME-targeting stimulatory or inhibitory protein is different compared to the antibody expressed by the virus provided by claim 1.   Further comprising providing the subject with at least one additional virus, wherein the at least one additional virus is: (i) two or more antibodies that target the tumor microenvironment (TME); (ii) Expresses two or more stimulatory or inhibitory proteins that target TME, or (iii) at least one antibody that targets TME and at least one protein that stimulates or inhibits TME; Two additional viruses are: (i) two or more antibodies that target the tumor microenvironment (TME); (ii) two or more stimulatory or inhibitory proteins that target TME, or (iii) Target TME and at least one antibody that targets TME different from that expressed by the virus provided by Expressing intense resistance or inhibition of at least one protein, the method according to claim 1.   A recombinant virus capable of infecting cells in a solid tumor, wherein: (i) two or more antibodies that target the tumor microenvironment (TME); (ii) two or more stimulatory or inhibitory targets that target TME Or (iii) at least one antibody that targets TME and at least one stimulatory or inhibitory protein that targets TME.   27. The recombinant virus of claim 26, which is an oncolytic virus.   28. The recombinant virus of claim 27, wherein the oncolytic virus is a vaccinia virus.   29. The recombinant virus of claim 28, which is a replication competent oncolytic vaccinia virus (VACV).   27. The recombinant virus of claim 26 that expresses two or more antibodies.   The two or more antibodies are: (i) an antibody that binds to a protein that stimulates angiogenesis and / or angiogenesis; (ii) epidermal growth factor receptor (EGFR), Her2 / C-neu, Her3 and Her4. 32. The antibody of claim 30, wherein the antibody binds to a receptor tyrosine kinase selected from the group consisting of: and (iii) an antibody that binds to a protein involved in the development of epithelial-mesenchymal interaction. Recombinant virus.   The following: 2 selected from the group consisting of an antibody that binds to vascular endothelial growth factor (VEGF), an antibody that binds to epidermal growth factor receptor (EGFR), and an antibody that binds to fibroblast activation protein (FAP) 32. The recombinant virus of any of claims 26-31, comprising two or more heterologous nucleic acids encoding one or more antibodies.   35. The recombinant virus of claim 32, wherein one of the two or more antibodies is an antibody that binds to VEGF.   34. The recombinant virus of claim 33, wherein the antibody that binds to VEGF is G6-31.   35. The recombinant vaccinia virus of claim 32, wherein one of the two or more antibodies is an antibody that binds to EGFR.   36. The recombinant virus of claim 35, wherein the antibody that binds to EGFR is anti-EGFR VHH.   33. The recombinant virus of claim 32, wherein one of the two or more antibodies is an antibody that binds to FAP.   38. The recombinant virus of claim 37, wherein the antibody that binds to FAP is M036.   35. The recombinant virus of claim 32, wherein the two or more antibodies comprise an antibody that binds to VEGF and an antibody that binds to EGFR.   35. The recombinant virus of claim 32, wherein the two or more antibodies include an antibody that binds to VEGF and an antibody that binds to FAP.   35. The recombinant virus of claim 32, wherein the two or more antibodies include an antibody that binds to EGFR and an antibody that binds to FAP.   30. The recombinant virus of claim 29, wherein the VACV is selected from GLV-1h444 and GLV-1h446.   The recombinant virus according to claim 26, which is a Lister strain.   29. The recombinant virus of claim 28, wherein the A34R gene is replaced with an A34R gene from another vaccinia virus strain.   45. The recombinant virus of claim 44, wherein the A34R gene is replaced with an A34R gene from vaccinia IHD-J strain.   29. The recombinant virus of claim 28 comprising a deletion of the A35R gene.   47. The recombinant virus according to any of claims 26 to 46, further comprising an additional heterologous nucleic acid molecule encoding a diagnostic or therapeutic protein.   48. The recombinant virus of claim 47, wherein the additional heterologous nucleic acid molecule encodes a diagnostic protein.   The diagnostic protein is selected from among luciferases, fluorescent proteins, iron storage molecules, iron transporters, iron receptors, or proteins that bind to an imaging agent, a chromophore, or a detectable compound or a detectable ligand. Item 49. The recombinant virus according to Item 48.   48. The recombinant virus of claim 47, wherein the additional heterologous nucleic acid molecule encodes a therapeutic protein.   The therapeutic protein is a cytokine, chemokine, immunomodulatory molecule, antigen, single chain antibody, antisense RNA, prodrug converting enzyme, siRNA, angiogenesis inhibitor, biological toxin, antitumor oligopeptide, mitotic inhibition 51. The recombinant virus of claim 50, selected from drug proteins, anti-mitotic oligopeptides, anti-cancer polypeptide antibiotics, and tissue factor.   52. A host cell comprising the recombinant virus according to any one of claims 26 to 51.   A tumor cell comprising the recombinant virus according to any one of claims 26 to 51.   52. A mammalian organism comprising or infected by the recombinant virus of any of claims 26-51.   52. Use of the recombinant vaccinia virus according to any of claims 26 to 51 for the treatment of a tumor in a subject.   52. Use of the recombinant virus according to any of claims 26 to 51 for the preparation of a pharmaceutical composition for the treatment of tumors in a subject.   57. Use according to claim 56, wherein the pharmaceutical composition further comprises an anticancer compound. Use of a cell comprising a recombinant virus capable of infecting cells in a tumor or a virus capable of infecting cells in a tumor, wherein the method for treating a solid tumor in a subject infects cells in said tumor. Administering to said subject a cell comprising a recombinant virus obtained or a virus capable of infecting cells in said tumor, wherein said virus targets: (i) two or more targeting the tumor microenvironment (TME) An antibody; (ii) expressing two or more proteins that stimulate or inhibit TME, and (iii) at least one antibody that targets TME and at least one protein that stimulates or inhibits TME To use.

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